In general, fluorescence involves the absorption of light (i.e., a photon or photons) by a molecule (i.e., a chromophore), thereby promoting the absorbing chromophore to an excited electronic state. After excitation by the incident photon, the chromophore typically undergoes an internal redistribution of energy, followed by a radiative decay back to the ground state. The internal redistribution of energy can be via a number of processes, such as, vibrational relaxation. Because energy is lost during the internal relaxation processes, the light released during the final radiative decay process is red-shifted to longer wavelengths.
The fluorescence process is an inelastic process characterized by a difference in the incident and the emitted wavelengths. The incident wavelength can be that of excitation or input light, and the emitted wavelength can be that of fluorescence or output light. FIG. 1A depicts an absorption and relaxation process of fluorescence.
Fluorescence techniques have been used as a method of cancer screening. Cancer remains one of the most lethal diseases in the world, and cancer screening and early interdiction are very important tools in reducing cancer-related deaths. Cervical cancer, colorectal cancer, oral cancer, and skin cancers strike millions each year. S. E. Waggoner, Cervical Cancer, Lancet, 361:2217-2225 (2003). With these cancer types in particular, the potential benefits of improved screening are enormous. These cancers are invariably preceded by dysplastic precancerous cellular changes, in which histological changes associated with malignancy are often confined to the epithelial layer. C. S. Herrington, M. Wells, Premalignant and malignant squamous lesions of the cervix, In H. Fox, M. Wells (eds), Haines and Taylor obstetrical and gynecological pathology, 5th edition, Edinburgh: Churchill Livingstone, pp. 297-338 (2002). Dysplasia, i.e., unequivocal neoplastic epithelium, is commonly relied on as a biomarker of malignancy. R. H. Riddell et al., Dysplasia in inflammatory bowel disease: standardized classification with provisional clinical implications, Hum. Pathol., 14:931-968 (1983).
While cancer screening is very important for early detection and treatment, many biologic processes exist that cannot be easily or directly monitored with visible microscopy. Additionally, these processes also cannot be easily or directly monitored using advanced analysis/imaging tools such as magnetic resonance imaging (MRI), computer tomography (CT), or nuclear imaging, due to the fact that key molecules in these processes are not distinguishable from each other via these methodologies. Due to these factors, direct visual observation, both externally and endoscopically, is generally used, including colorectal screening, gastric and dermatological applications. However, while the human eye can process many visual cues, several of the early molecular changes associated with cancer are simply not discernable by even the most skilled practitioners. For example, routine endoscopy for colorectal cancer screening has a miss rate of up to 24%, with substantially higher figures when the lesions are flat. D. K. Rex et al., Colonoscopic miss rates of adenomas determined by back-to-back colonoscopies. Gastroenterology, 112:24-28 (1997); B. J. Rembacken et al. Flat and depressed colonic neoplasms: a prospective study of 1000 colonoscopies in the UK, Lancet, 355:1211-1214 (2000).
In response to these shortcomings, several other techniques have been developed for cancer and precancerous evaluation. D. Benaron, D. Stevenson, Optical time-of-flight and absorbance imaging of biologic media, Science, 259:1463-1466 (1993); B. Chance, Near-infrared images using continuous, phase-modulated, and pulse light with quantitation of blood and blood oxygenation. Ann. NY Acad. Sci., 838:29-45 (1993); R. Alfano et al., Advances in optical imaging of biomedical media, Ann. NY Acad. Sci., 820:248-270 (1997); S. Andersson-Engels et al., In vivo fluorescence imaging for tissue diagnostics, Phys. Med. Biol., 42:815-824 (1997); P. Contag et al., Bioluminescent indicators in living mammals, Nature Med., 4:245-247 (1998); J. C. Hebden, D. T. Delpy, Diagnostic imaging with light, Br. J. Radiol., 70:S206-S214 (1997); R. Manoharan et al., Raman spectroscopy and fluorescence photon migration for breast cancer diagnosis and imaging, Photochem. Photobiol., 67:15-22 (1998); G. Tearny et al., In vivo endoscopic optical biopsy with optical coherence tomography, Science, 276:2037-2039 (1997).
Fluorescence-based cancer imaging and detection makes use of the fluorescence characteristics of naturally occurring molecules such as collagen, nicotinamide adenine dinucleotide, flavins, and porphyrins, that is, the study of natural fluorescent compounds. Such systems are said to be characterized by auto-fluorescence. In contrast, the addition of compounds to the tissue or cellular system (i.e., exogenous fluorophores) may be performed with a goal of preferential accumulation in the neoplastic tissue. C. Arens et al., Indirect fluorescence laryngoscopy in the diagnosis of precancerous and cancerous laryngeal lesions, Eur. Arch. Otorhinolaryngol, 264:621-626 (2007). It is known that malignant tumors can accumulate endogenous fluorophores (i.e., auto-fluorescence). A. Policard, Etude sur les aspects offerts par des tumours experimentales examinees a la lumiere de Wood, CR Soc. Biol., 91:742-743 (1924). Additionally, benign and malignant tumors may have differences that can be detected by fluorescence techniques. R. Alfano et al., Laser induced fluorescence spectroscopy from native cancerous and normal tissue, IEEE J. Quantum Electron, 20:284-291 (1984).
Fluorescence spectroscopy has been used to attempt to diagnose cervical intraepithelial neoplasia (CIN), an important dysplastic precancerous change in which histological changes are confined to the epithelial layer. A. Mahadevan et al., Study of the fluorescence properties of normal and neoplastic human cervical tissue, Lasers Surg. Med., 13:647-655 (1993). In addition, endoscopic detection of gastrointestinal cancers and diseases is an excellent example of a clinical application of fluorescence spectroscopy for real-time, non-invasive detection of dysplasia and early cancer. C. Arens et al., Indirect fluorescence laryngoscopy in the diagnosis of precancerous and cancerous laryngeal lesions, supra.Eur. Arch. Otorhinolaryngol, 264:621-626 (2007); J. Haringsma, G. N. J. Tytgat, Fluorescence and autofluorescence, Bailliere's Clinical Gastroenterology, 13:1-10 (1999). Other important applications for auto-fluorescence techniques include colorectal cancer screening, oral oncology, and in vivo imaging of enzyme activity. D. P. Hurlstone, S. Brown, Techniques for targeting screening in ulcerative colitis, Postgrad. Med. J., 83:451-460 (2007); B. W. Chwirot et al., Spectrally resolved fluorescence imaging of human colonic adenomas, J. Photochem. Photobiol. B: Biol., 50:174-183 (1999); D. C. G. De Veld et al., The status of in vivo autofluorescence spectroscopy and imaging for oral oncology, Oral Oncology, 41:117-131 (2005); M. A. Funovics et al., Catheter-based in vivo imaging of enzyme activity and gene expression: feasibility study in mice, Radiology, 231:659-666 (2004).
In addition to fluorescence as described above, other optical techniques include Raman spectroscopy and multi-photon fluorescence. Raman spectroscopy is a inelastic light scattering process in which incident photons either add (Stokes shift) or subtract (Anti-Stokes shift) energy to the vibrational energy of the host material, resulting in an energy shift of the subsequently scattered photon. Because Raman scattering (i.e., Raman spectroscopy) is sensitive to the local molecular structure, it has been used as an optical sensing technique, including for cancer detection. K. Sokolov et al., Optical systems for in vivo molecular imaging of cancer, Tech. in Cancer Res. & Treatment, 2:491-504 (2003); U. Utzinger et al., Near-infrared Raman spectroscopy for in vivo detection of cervical percancers, Appl. Spectroscopy, 55:955-959 (2001). Multi-photon fluorescence uses a combination of two or more photons to excite the target molecular structure, after which typical fluorescence processes, as discussed above, occur. By using multiple photons of longer wavelength, better imaging (notably biological imaging) can be obtained by eliminating problems such as matrix attenuation of light related to, for example, shorter wavelength, single-photon fluorescence, out-of-plane fluorescence, and/or photobleaching. D. J. Stephens, et al., Light microscopy techniques for live cell imaging, Science, 300:82-86 (2003).
Clinical applicability of fluorescence, and other optical techniques described above, has been limited by large patient-to-patient variations in fluorescence properties, as well as non-uniform uptake and distribution of exogenous fluorescence agents and biomarkers. C. Arens et al., Indirect fluorescence laryngoscopy in the diagnosis of precancerous and cancerous laryngeal lesions, supra.Eur. Arch. Otorhinolaryngol, 264:621-626 (2007); S.F. Martin et al., Fluorescence spectroscopy of an in vitro model of human cervical precancer identifies neoplastic phenotype, Int. J. Cancer, 120:1964-1970 (2007); C. Brookner et al., Effects of biographical variables on cervical fluorescence emission spectra, J. Biomed. Optics., 8:479-483 (2003). While fluorescence and Raman techniques have been a valuable source of diagnostic information for detecting precancerous tumors, many drawbacks still exist in the art. The development of improved methods for cancer screening could help increase the percentage of early detection and curable cancer cases. Similarly, the addition of further functionality to biological imaging schemes (e.g., fluorescence and multi-photon) is also desirable.